Sex-specific movement ecology of the shortest-lived tetrapod during the mating season

Sex-specific reproductive strategies are shaped by the distribution of potential mates in space and time. Labord’s chameleon (Furcifer labordi) from southwestern Madagascar is the shortest-lived tetrapod whose life-time mating opportunities are restricted to a few weeks. Given that these chameleons grow to sexual maturity within about three months and that all individuals die soon after breeding, their mating strategies should be adapted to these temporal constraints. The reproductive tactics of this or any other Malagasy chameleon species have not been studied, however. Radio-tracking and observations of 21 females and 18 males revealed that females exhibit high site fidelity, move small cumulative and linear distances, have low corresponding dispersal ratios and small occurrence distributions. In contrast, males moved larger distances in less predictable fashion, resulting in dispersal ratios and occurrence distributions 7–14 times larger than those of females, and males also had greater ranges of their vertical distribution. Despite synchronous hatching, males exhibited substantial inter-individual variation in body mass and snout-vent length that was significantly greater than in females, but apparently unrelated to their spatial tactics. Females mated with up to 6 individually-known mates, but frequent encounters with unmarked individuals indicate that much higher number of matings may be common, as are damaging fights among males. Thus, unlike perennial chameleons, F. labordi males do not seem to maintain and defend territories. Instead, they invest vastly more time and energy into locomotion for their body size than other species. Pronounced variation in key somatic traits may hint at the existence of alternative reproductive tactics, but its causes and consequences require further study. This first preliminary study of the mating system of a Malagasy chameleon indicates that, as in other semelparous tetrapods, accelerated life histories are tied to a mating system with intense contest and scramble competition among males.

www.nature.com/scientificreports/ This phenomenon is driven to the extreme by a species from the deciduous dry forests of south-western Madagascar. Labord's chameleons (Furcifer labordi, Grandidier, 1872) spend more of their lifespan as an embryo than as a developed chameleon so that for parts of the year there are only eggs, but no chameleons in the forest; a life history that is unparalleled among tetrapods 10 . Their unique semelparous life history is complemented by a brief reproductive season, in which male-male encounters are more physically intense than in perennial congeners, and females have a longevity advantage similar to that observed in semelparous marsupials [21][22][23] . Mating behavior appears to be not only determined by male-male competition, because the length of the fleshy rostral appendage of males appears to be evaluated by female mate choice 24 . Observations of vast daily movements (based on unpublished radiotracking data 21 ), costly male-male encounters, and mate guarding of solitary females during an extremely short period of reproductive activity raise questions about the optimal male reproductive strategy under these conditions 25 .
Due to their cryptic nature and small to moderate size, studies on the social organization and mating system of chameleons are generally scarce. Radiotelemetry studies have focused primarily on aspects of habitat use, e.g. in invasive East African (Trioceros sp.) and Malagasy (Furcifer sp.) chameleons in Hawaii and Florida [26][27][28] and in South African dwarf chameleons (Bradypodion sp.) in semi-urban habitats 29 . Only the mating system of perennial European chameleons (Chamaeleo sp.) have been studied in any detail [30][31][32][33] . While these studies are informative about the mating systems and spatial strategies of ' conventional' chameleons, they offer no basis for predicting reproductive strategies in F. labordi, however, because the latter's entire life span is shorter than the age of sexual maturity in many other chameleons, and differences in size among males are much reduced compared to longer-lived species 21 . Moreover, the only currently available information on mating behavior of F. labordi is based on observations in staged arena trials 21,24 . We present here the first results of a radiotracking study of wild F. labordi during their brief (< 4 weeks) annual mating season, combining spatial and behavioral data for a first characterization of this species' mating system. Despite its limited duration and sample size resulting from the challenges of finding and following large numbers of males and females in a complex tropical forest habitat, our study provides a first characterization of the key features of the mating system of a Malagasy chameleon.

Results
Body size and condition. Female F. labordi had a mean body mass of 8.7 g (range 5.5-12.5 g, N = 37) and a mean snout-vent-length of 71.7 mm (range 64.5-80.1 mm, N = 36), while male F. labordi had a mean body mass of 16.2 g (range 10.5-24.5 g, N = 24) and a mean snout-vent-length of 92.6 mm (range 75.9-104.9 mm, N = 24). The rostral appendage of male F. labordi (N = 22) had a mean height of 5.5 mm (range 4.1-6.7 mm), a mean width of 1.6 mm (range 1.1-2.0 mm) and a mean length of 13 mm (range 9.1-17.2 mm). The mean height of the male cranial casque was 10.9 mm (range 6.8-14.8 mm). Body mass and snout-vent-lengths within male F. labordi showed greater variability than within females, with body mass of males detected in the same week differing by up to 110% and snout-vent-length differing by up to 30% (F-test; body mass: F = 0.09, p = 0.005, snoutvent-length: F = 0.21, p = 0.03). Variance in body condition residuals was also larger among males (range − 0.22 to 0.31) than among females (range − 0.14 to 0.09, F test: F = 0.29, p = 0.07).
Body condition and secondary sexual characteristics were not obviously correlated to movement strategies, with the smallest males covering similar 5-day occurrence distributions, cumulative distances and dispersal ratios as the largest males (e.g. M81, initial mass 10.5 g, initial SVL 75.9 mm, five-day-MCP 1860 m 2 , 5-day-cumulative distance 172 m, 5-day-DR 0.21 versus M90, initial mass 18.5 g, initial SVL 97.3 mm, 5-day-MCP 1609 m 2 , 5-daycumulative distance 166 m, 5-day-DR 0.25). In females, body condition and dispersal ratio (DR) appeared to be positively correlated in the 5-day data set (rs: 0.9, p = 0.08, n = 5), but were negatively correlated in the full data set (rs: − 0.7, p = 0.04, N = 9), indicating that the sample size was too small to obtain biologically meaningful results.

Ranging.
A total of 39 Furcifer labordi (21 females and 18 males) were equipped with radio-transmitters.
Of these, 12 females and 9 males lost their transmitters after less than 3 days, i.e., a maximum of 9 relocations. For the remaining 9 females and 9 males, the median number of relocations was 23 (range 10-37) and 26 (range 11-69), respectively, and data from these individuals collected throughout the mating season were used for occurrence distribution and movement trajectory analyses. An additional 14 males and one female were detected in the study area, but roosted too high in the canopy to be captured, illustrating high local densities and indicating that observations on radio-tracked individuals only represent a fraction of individuals and interactions in the habitat at any time ( Fig. 1). More precise estimates of the total number of males and females in the study area were not available, however, because of visual constraints and a trade-off between censusing and radiotracking during the extremely short mating season.
As these values were derived from unequal tracking periods, we assessed site fidelity for a standardized timespan of 5 days, and calculated a dispersal ratio (DR) by dividing the Euclidean distance through the cumulative distance an individual had travelled in this period. On five consecutive days, females moved a median cumulative distance of 84 m (range 65-123, n = 5), whereas males moved a median cumulative distance of 158 m (range  Fig. 2A).
Median roosting height of females was 205 cm (range 14-769 cm), while males had a median roosting height of 270 cm above ground (range 4-1062 cm). Among the 18 tracked individuals for which multiple measurements were available, individual range of vertical distribution, i.e. range from lowest to highest recorded height above ground, was higher in males (median 467 cm, range 170-1014, N = 9) than in females (median 250 cm, range 87-565, N = 9; Fig. 2B), indicating that males are more mobile in the third dimension as well.
Mating system. Due to low transmitter retention, difficult observation conditions and an unknown number of additional individuals present in the study area, the following accounts of mating behavior should be considered as preliminary. One male (M81) traversed the areas utilized by 10 radio-tracked females, for 7 of which occurrence distribution estimates are available and 3 of which were located 4-7 times. It was observed courting or mate guarding 6 of these females, and engaged in altercations with other known males pursuing the same females at least 3 times. The occurrence distributions of the females sequentially mated by this male during this period of ca. 3 weeks were traversed by 10 known radio-tracked males during this period, and an additional 6 unmarked males were observed in the area. Four other tracked males and one unmarked male either mate guarded or stayed close to the same females as M81 during the same period, and encounters between males occurred at least 3 times. All males showed erratic movement trajectories with both rapid 'bursts' interspersed by longer periods with little or no movement. These observations suggests that other male F. labordi, even though their interactions with known and unknown conspecifics in the habitat were often impossible to detect, pursued a similar strategy of rapid sequential mating attempts.
Different males were repeatedly tracked at relatively small distances to each other (< 10 m), and also always in close vicinity to at least one known female's location. It is unclear, however, whether fights between these individuals took place, whether they were aware of each other (and potentially additional, non-tracked males), and whether some of the large distances traversed by males between tracking locations were due to displacements by other males. In two instances, two different males slept in direct vicinity of each other (< 3 m and < 5 m distance, www.nature.com/scientificreports/ respectively) and close to a nearby female. Several tracked males showed bite marks at the tail base or abrasions at the rostral appendage and jaw, likely a result of 'jawlocking' and biting during combat. Of the 21 radio-tracked females, seven deposited eggs during the period of tracking, and three clutches containing 6-8 eggs were recovered. However, because the timing of fertilization could not be determined for all females, it was impossible to analyze female ranging behavior as a function of reproductive status. Six females entered abandoned terrestrial ant burrows (of the genus Camponotus) for egg deposition. Of those, one deceased within 12, and another one within ca. 48 h after egg deposition, highlighting the large energetic investment into reproduction in this species.

Discussion
By radiotracking male and female F. labordi during their short annual reproductive activity, this study provided first baseline data on the sex-biased movement ecology and underlying mating strategies of the shortest-lived tetrapod. Our data indicated that females exhibited high site fidelity, with low cumulative and linear distances moved, and corresponding low dispersal ratios and small occurrence distributions. In contrast, males moved larger distances in less predictable fashion, resulting in higher dispersal ratios and occurrence distributions 7-14 times larger than those of females. Consequently, their occurrence distributions encompassed those of several females, with males apparently searching for and visiting one female after another, interrupted by agonistic interactions and displacement when encountering male rivals.
Low site fidelity, small distances between individual center points, and strong overlap of occurrence distributions in males suggest that, unlike in perennial European chameleons 33 , F. labordi males do not maintain and defend territories. This notion is further supported by anecdotal evidence: one male traversed 10 and mateguarded or courted at least 6 known females within a period of 21 days, and it encountered other radio-tracked males attempting to breed with the same females at least 3 times. As additional males were regularly observed in this area, and as further individuals of both sexes likely remained completely undetected during the duration of the study, the true number of females courted per male, courting males per female, and of agonistic male-male encounters is likely even higher. In combination, these observations suggests that F. labordi has a polygynandrous mating system with pronounced contest and scramble competition among males. Below, we will compare the movement ecology of F. labordi to what is known from longer-lived relatives, and will discuss its association with life-history traits, reproductive strategies, and the resulting mating system.

Movement ecology.
Occurrence distributions estimated in the present study are larger than estimates reported for larger and longer-lived chameleon species 33,34 . Male European chameleons (C. chamaeleon) utilized 100% MCPs ranging from 40 to 719 m 2 during their reproductive season 33 (Cuadrado 2001), averaging ca. 1/10th of 95% MCPs found for male F. labordi. It is therefore apparent that male F. labordi cover vastly larger areas than the only other chameleon species for which movement data during the reproductive season exist. Considering the smaller body size of F. labordi, males of this species therefore appear to invest proportionally more time and energy into locomotion than their European relatives.
Movement strategies of F. labordi clearly differ between the sexes and provide insights into their mating system. Male-biased movement metrics have been associated with polygynous mating systems in mammals 35 . The estimates of movement generated here (combined with the observations on the mating behavior of F. labordi) are strikingly congruent with this notion, perhaps with the exception that female defense appears to be neither beneficial nor feasible in this extremely short-lived species. Nonetheless, we cannot exclude the possibility with the presently available preliminary data that some males patrolled an area that contained multiple females whereas others, notably smaller males, used a sneaker strategy that was also associated with high locomotor activity.
Male-biased increased movement as a mate-finding strategy has been suggested for several vertebrate species [36][37][38] . For example, males with larger home ranges had access to more potential mates in speckled rattlesnakes (Crotalus mitchellii), where strong competition arises from the clumped spatiotemporal distribution of receptive females 38 . Investment in presumably costly mate searching appears to be the driver of male-biased movement in F. labordi as well. The observed variation in movement estimates among different males may therefore be indicative of the number of females encountered. Because movement estimates were not linked to metrics of body condition or secondary sexual characters, it remains unclear why some males moved less than others, however, without displaying obvious territoriality or mate defense. Thus, there is variation in male movement ecology that remains to be explained.
Movement of female F. labordi was much more confined than that of males, indicating that roaming is less beneficial for females. High site fidelity might instead be beneficial when resources are locally abundant and do not exhibit spatio-temporal variation, a scenario that applies to the deciduous forest habitat of these chameleons only during the short rainy season. As feeding resources and small-scale thermal gradients appeared to be at no shortage (e.g. in almost 50% of direct observations, females were feeding), females might not need to move large horizontal distances, and can apparently sustain themselves in small and even overlapping home ranges. Indeed, large movements were exclusively observed in gravid females, apparently searching for suitable egg deposition sites, but the limited available data did not permit a formal analysis of the effects of female reproductive state on their ranging behavior because the exact dates of mating were not known for most females. The divergent mate acquisition strategies of males and females appear therefore not to be driven by limited feeding resources and structural or thermal requirements. Instead, males engage in movement to increase their number of encounters with females, at the cost of increased predation risk and encounters with competitors. Females, in contrast, might face no limiting resource other than suitable egg deposition sites, rendering them more philopatric. www.nature.com/scientificreports/ Life history traits. Given the prevalence of male-male combat and female choice reported for this species 21,24 , the sex differences in secondary sexual characters observed here are to be expected. However, interindividual variation of up to 110% in body mass and 30% in snout-vent-length among individuals present at the same time was unexpected, considering the synchronous hatching of F. labordi. With the short period of female receptivity being the limiting factor, males growing and maturing at the fastest rate should enjoy the greatest mating success, if male size confers reproductive advantages. The observed pronounced variation in male body condition might therefore be the result of a male arms race, but it remains unknown which factors allow or limit growth to particularly large or small sizes, respectively. There is experimental support for the importance of male body size in both intra-and intersexual selection 21 , but it remains unknown how frequent and under which circumstances male-male and male-female encounters occur under wild conditions, and which fitness consequences body condition and secondary sexual characteristics have for individual reproductive success. Besides, 35% of experimental male-male altercations were won by the smaller competitor 21 , highlighting that additional factors play a role in intrasexual selection. Small males ought to have a substantial disadvantage concerning mating opportunities and success 32 , unless they follow a sneaker strategy. However, data from the single male for which a detailed account of mate guarding and mating attempts could be compiled contradicts this notion: it was the smallest of all radio-tracked males in terms of both SVL and body mass when first equipped with a transmitter (first record: 76 mm, 10.5 g), and even though it grew during the study (last record: 84.2 mm SVL, 11.5 g), it never reached nearly the size of some other males in the same area at the same time (e.g. last records of 103.5 mm SVL, 23.5 g or 104.9 mm, 20 g). While the outcome of male-male altercations and mating attempts of this male remain unknown, it pursued the same movement strategy as vastly larger males. Additional data are now required to determine whether males of different size pursue alternative male reproductive tactics 39 .

Mating system. Because most previous chameleon studies of processes involved in accessing and choosing
mates have been conducted with animals in permanent or temporary captivity [14][15][16][17]24 , empirical data on chameleon mating systems are scarce [30][31][32][33][34]40,41 . While it has been suggested that they possess a wide range of different mating systems 40 , the clade is generally assumed to be polygamous in the sense that "(m)ales may mate with more than one female and females may mate with different males during the same or different ovarian cycles" 40 when given the opportunity. Whether females mate with one or multiple males within one ovarian cycle is likely dependent on encounter rates and male monopolization potential. Studies in captivity indicated that the dynamics of male color change may impact female mate choice 42 and predict the outcome of male-male agonistic interactions 43 , but the relative importance of these processes in shaping the mating system of wild (panther) chameleons remains unknown. Our study is therefore the first to contribute observations on the mating system of a wild population of Malagasy chameleons.
Compared to perennial chameleons, in which some males are able to monopolize the most fecund females for up to 46 days 32 , male monopolization potential in F. labordi appears to be much reduced, but additional observations of matings are required to better assess the strategies males use to defend their reproductive success. Some populations of Canadian garter snakes (Thamnophis sirtalis) also have just a few weeks to mate after larger numbers of spatially clumped females emerge from communal overwintering dens. They mate promiscuously but males with better body condition are more successful and use mating plugs rather than mate guarding or extended mating to secure their reproductive success 44 . More comparative data on male reproductive tactics in other reptilian taxa with short breeding periods are required for a more detailed appraisal of the available options.
While clutch size is strongly correlated to female body size in chameleons 45 , it is unlikely that variation in female F. labordi fecundity is similarly larger as in the larger perennial congeners, for which estimates of 10-30 (F. cf. nicosiai) and an average of 42 eggs (F. oustaleti) have been reported 23 . Three females during this study had clutches containing 6, 7 and 8 eggs respectively, indicating some variation in fecundity among female F. labordi, but we found no evidence for a decreasing size-rank-order of male mate guarding, as observed in perennial European chameleons 33 . As female squamates are able to store sperm internally, and therefore can control the timing of fertilization and possibly paternity 46,47 , male mating success does not guarantee fertilization success. It is therefore in the interest of males to maximize both copulation duration per female and the number of mates. Since the vast majority of female F. labordi produce only a single clutch during their lives 23 , mating polyandrously might reduce the risk of producing unfertilized eggs and improve genetic diversity of their offspring 48 .
In contrast, males can generally increase their reproductive success by mating with additional females. As only about 5% of females in F. labordi experience a second ovarian cycle 23 , the operational sex ratio changes drastically as the breeding season advances, which may explain the parallel increase in male agonism 49 . With increasingly male-biased operational sex ratios, levels of polyandrous matings and multiple paternity ought to increase as well 50 , but future studies are required to test these predictions. Larger sample sized would also permit studies of the effects of sex ratio dynamics on male ranging behavior.

Conclusions
This work represents the first study of the mating system of a Malagasy chameleon, and provides first insight into the movement ecology and mating system of the shortest-lived tetrapod. Perhaps due to low variation in female fecundity, and both spatial and temporal factors that impede the formation and defense of stable territories, there appears to be neither an incentive nor an opportunity for male F. labordi to monopolize receptive females. Observed variation in movement patterns, body size and secondary sexual characters were not indicative of alternative reproductive strategies among males, but additional studies of individually known males across their entire life span will be required to identify potential causes and behavioral consequences of striking variation in male body size. Sex differences in movements in all 3 dimensions indicate the prevalence of a polygynandrous www.nature.com/scientificreports/ mating system, characterized by pronounced male contest and scramble competition and female polyandry. The mating system of F. labordi therefore resembles that of other semelparous tetrapods 51 , suggesting that accelerated life histories are tied to particular mating systems.

Methods
Study site. The study was conducted in Kirindy Forest (44°39′E, 20°03′S, ca. 30-60 m asl), which is located ca. 60 km north of the coastal town of Morondava and ca. 20 km inland from the Mozambique channel in the central Menabe region of western Madagascar. The climate in this area is characterized by a short, hot and wet austral summer between November and March, and a cooler austral winter with little or no rainfall 52 . The study area, locally known as CS7, is a 1 km 2 forest block intersected by the Kirindy River and covered by a grid system of foot trails 25 m apart that allow orientation and movement through the dense forest.
Radio-tracking of Furcifer labordi. Between 23 January 2020 and 09 March 2020, male and female F. labordi were searched for and equipped with small radio-transmitters (BD-2, 0.64 g, 151 MHz, Holohil Systems Ltd.), using a cyanoacrylate adhesive (UHU 'superglue flex gel' , Bolton adhesives). The transmitter weighed less than 7.4% of the average female mass and less than 4% of the average male mass. The study had to be terminated due to a Covid-related evacuation. Individuals were searched for along the grid system between 18:30 and 22:00 h, using a Fenix HL60R LED headlamp. Upon detection, their roosting height above ground was measured (using a Bosch PLR50 laser rangefinder), as well as their body temperature at the center of the lateral body surface (using a Laserliner ThermoSpot infrared thermometer). Chameleons were then captured to measure their snout-vent-length (SVL) and, in males, two secondary sexual characters to the nearest 0.1 mm with digital calipers: The height, width and length of the rostral appendage as well as the height of the cranial casque. Body mass was then measured using a Pesola spring balance (60 g, d = 0.5 g). Chameleons were immobilized in a piece of soft cloth and the transmitter was attached above the left or right hip of the animal, taking advantage of their dorso-laterally flattened body. Curing of the adhesive took between 4 and 10 min. Each transmitter carried the two-digit serial number that was often visible with binoculars and also served as the animal ID. At the initial encounter and during all subsequent visual encounters with tracked individuals, a photograph of the animal was taken. After controlling the correct fit of the transmitters, the animals were released onto the same branch they were collected from, and a GPS coordinate was taken, using a Garmin eTrex 20x. To facilitate subsequent tracking of the animals, the location was marked using a colored flag.
Chameleons were tracked using a Telonics TR-4 receiver and F150-151-3FB antenna. Each individual was located three times per day: between 07:00 and 10:00 h, between 12:00 and 15:00 h, and between 18:30 and 21:30 h, covering both diurnal activity patterns and nocturnal roosting site choice. Conventional triangulation methods were used, facilitated by the grid system. Upon visual detection of an individual, its height above ground and body temperature were measured as described above. Focal individuals were observed for 2-5 min, and their current activity (sleeping, resting, feeding, moving, interaction with conspecifics, i.e., mate guarding, courtship or agonistic behavior, other) was noted. The GPS coordinate was then taken, and the location of the animal was marked. Due to the dense and sometimes impenetrable undergrowth and the rich foliage during rainy season, it was not always possible to establish visual contact with the tracked individual, especially when the animals were active high in the canopy. In these cases, the GPS coordinate of the shrub or tree with the strongest signal was taken and marked. Data analyses. Data storage and exploration was conducted in Excel v16.0. Statistical analyses were conducted in R v4.0.2 53 using RStudio v1.3.959 54 . The packages plyr 55 , dplyr 56 and lubridate 57 were used for data wrangling, and the packages ggplot2 58 and cowplot 59 were used for the creation of graphs. The packages psych 60 , modelr 61 and car 62 were used to perform descriptive statistics. The package brunnermunzel 63 was used to perform nonparametric tests of stochastic equality for two samples with unequal variances in non-normally distributed data. We used the residuals of the linear regression between (log-transformed) body mass and SVL to operationalize body condition. Spearman's rank correlation tests were conducted to test for relationships between standardized movement metrics and measurements of body size, body condition, and measurements of secondary sexual characteristics. To assess the areas utilized by radio-tracked individuals, relocation data were used to estimate occurrence distributions. Terminology in space-use studies has often been ambiguous 64 , and this term was proposed recently 65 , replacing the more commonly used term home range 66 when it is not applicable.
The package amt 67 was used for the calculation of minimum convex polygon (MCP), local convex hull (k-LoCoH) and kernel-density-estimation (KDE) estimates of occurrence distributions, and for the analysis of simplified movement trajectories. Other geospatial analyses and the compilation of maps were conducted in QGIS v3.10.0 68 . Using the MCP method, the smallest polygon including all GPS points with interior angles less than 180 degrees was constructed. To exclude outliers, MCP estimates are commonly truncated to include only 95% of all available relocations for one animal. In addition, 50% MCPs were calculated to identify core areas of utilization. While MCPs are the classical and most intuitive method for estimating home range size, they have the tendency to include areas that are not utilized by the animal at all, and by default, their edges can only be of convex shape. Some of these limitations are avoided using the k-LoCoH method 69 , where MCPs, here called local convex hulls, are constructed for local subsets of each point in the dataset and its nearest neighbors. The resulting hulls are then merged from smallest (containing most locations) to largest (containing least locations) and can then be divided into isopleths containing a certain percentage of the whole data set. Unlike MCPs, LoCoHs allow for concavity and 'holes' in the estimated area. To compare the two methods, LoCoH estimates were also constructed at a 95% level, and, as the method is particularly suited to assess core areas, at a level of 50%. The KDE method 70 is a modelling approach that fits a probabilistic function to each data point, resulting in a surface

Funding
Open Access funding enabled and organized by Projekt DEAL. This research was funded by a research grant by the Association for the Study of Animal Behaviour to PMK and by a PROMOS scholarship to LH.